Though HIV-1 superinfection (SI) in an individual whose immune system may already have some immune deficiencies caused by the HIV infection does not truly represent the consequences of vaccination in an HIV-1 negative person, immunological and virological differences between superinfected and non- superinfected individuals or events occurring around the time of SI could help us to better understand protective immune responses and guide rational HIV vaccine design and development. Previous studies looking at the role of both cellular and humoral immune responses in HIV SI have provided conflicting results on their role is preventing SI. In addition no previous study has been able to identify SI in known partners with unlinked viruses where both immune responses and virus are characterized before and after SI. We want to contribute by analysing samples being collected from a large cohort of subtype A and D infected individuals within high risk cohorts where earlier studies show high rates of SI. We propose to identify linked SI among concordant positive couples using already stored specimens from an on-going cohort study. We will in addition employ very novel assays to detect SI and base our immunology studies on using autologous peptides and viruses. In addition, to our knowledge, no studies have examined the role of mucosal and innate immune responses in protection against SI, a gap this application proposes to fill. The role of IFN has been recently implicated in the transmission bottleneck; whether this has a role in SI need to be explored. There is an opportunity to investigate whether SI viruses have properties similar to founder viruses transmitted during primary infection in relation to IFN-resistant information that could lead to therapeutic and prophylactic interventions In these studies we will test the hypothesis that insufficient or low levels of both innate and adaptive immune responses, particularly neutralizing antibodies and binding antibodies to the viral envelope, are found in individuals prior to HIV-SI when compared to matched individuals who do not become superinfected. This will be a case control study to investigate the effect of exposures / protective factors such as neutralizing antibodies and T-cell subsets. For certain other assays further exploratory tests to generate hypotheses will be carried out on a sub-sample of the participants. The Specific Aims are: 1) Identify HIV SI in ongoing high risk populations and in HIV infected couples with unlinked infections. 2) Examine anti-HIV antibody and CD8 T cell responses in matched SI case-controls of high risk individuals and in linked couples before and after SI 3) Explore if circulating and/or mucosal innate immune responses protect against HIV-1 superinfection